Chromatin Folding, Fragile Sites, and Chromosome Aberrations Induced by Low- and High- LET Radiation

We previously demonstrated non-random distributions of breaks involved in chromosome aberrations induced by low- and high-LET radiation. To investigate the factors contributing to the break point distribution in radiation-induced chromosome aberrations, human epithelial cells were fixed in G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome in separate colors. After the images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multimega base pair scale. Specific locations of the chromosome, in interphase, were also analyzed with bacterial artificial chromosome (BAC) probes. Both mBAND and BAC studies revealed non-random folding of chromatin in interphase, and suggested association of interphase chromatin folding to the radiation-induced chromosome aberration hotspots. We further investigated the distribution of genes, as well as the distribution of breaks found in tumor cells. Comparisons of these distributions to the radiation hotspots showed that some of the radiation hotspots coincide with the frequent breaks found in solid tumors and with the fragile sites for other environmental toxins. Our results suggest that multiple factors, including the chromatin structure and the gene distribution, can contribute to radiation-induced chromosome aberrations

WRN participates in translesion synthesis pathway through interaction with NBS1.

Werner syndrome (WS), caused by mutation of the WRN gene, is an autosomal recessive disorder associated with premature aging and predisposition to cancer. WRN belongs to the RecQ DNA helicase family, members of which play a role in maintaining genomic stability. Here, we demonstrate that WRN rapidly forms discrete nuclear foci in an NBS1-dependent manner following DNA damage. NBS1 physically interacts with WRN through its FHA domain, which interaction is important for the phosphorylation of WRN. WRN subsequently forms DNA damage-dependent foci during the S phase, but not in the G1 phase. WS cells exhibit an increase in spontaneous focus formation of poleta and Rad18, which are important for translesion synthesis (TLS). WRN also interacts with PCNA in the absence of DNA damage, but DNA damage induces the dissociation of PCNA from WRN, leading to the ubiquitination of PCNA, which is essential for TLS. This dissociation correlates with ATM/NBS1-dependent degradation of WRN. Moreover, WS cells show constitutive ubiquitination of PCNA and interaction between PCNA and Rad18 E3 ligase in the absence of DNA damage. Taken together, these results indicate that WRN participates in the TLS pathway to prevent genomic instability in an ATM/NBS1-dependent manner

Cellular responses to DNA double-strand breaks after low-dose γ-irradiation

DNA double-strand breaks (DSBs) are a serious threat to genome stability and cell viability. Although biological effects of low levels of radiation are not clear, the risks of low-dose radiation are of societal importance. Here, we directly monitored induction and repair of single DSBs and quantitatively analyzed the dynamics of interaction of DNA repair proteins at individual DSB sites in living cells using 53BP1 fused to yellow fluorescent protein (YFP-53BP1) as a surrogate marker. The number of DSBs formed was linear with dose from 5 mGy to 1 Gy. The DSBs induced by very low radiation doses (5 mGy) were repaired with efficiency similar to repair of DSBs induced at higher doses. The YFP-53BP1 foci are dynamic structures: 53BP1 rapidly and reversibly interacted at these DSB sites. The time frame of recruitment and affinity of 53BP1 for DSB sites were indistinguishable between low and high doses, providing mechanistic evidence for the similar DSB repair after low- and high-dose radiation. These findings have important implications for estimating the risk associated with low-dose radiation exposure on human health

Dynamin impacts homology-directed repair and breast cancer response to chemotherapy

After the initial responsiveness of triple-negative breast cancers (TNBCs) to chemotherapy, they often recur as chemotherapy-resistant tumors, and this has been associated with upregulated homology-directed repair (HDR). Thus, inhibitors of HDR could be a useful adjunct to chemotherapy treatment of these cancers. We performed a high-throughput chemical screen for inhibitors of HDR from which we obtained a number of hits that disrupted microtubule dynamics. We postulated that high levels of the target molecules of our screen in tumors would correlate with poor chemotherapy response. We found that inhibition or knockdown of dynamin 2 (DNM2), known for its role in endocytic cell trafficking and microtubule dynamics, impaired HDR and improved response to chemotherapy of cells and of tumors in mice. In a retrospective analysis, levels of DNM2 at the time of treatment strongly predicted chemotherapy outcome for estrogen receptor-negative and especially for TNBC patients. We propose that DNM2-associated DNA repair enzyme trafficking is important for HDR efficiency and is a powerful predictor of sensitivity to breast cancer chemotherapy and an important target for therapy

PTIP associates with Artemis to dictate DNA repair pathway choice.

PARP inhibitors (PARPis) are being used in patients with BRCA1/2 mutations. However, doubly deficient BRCA1(-/-)53BP1(-/-) cells or tumors become resistant to PARPis. Since 53BP1 or its known downstream effectors, PTIP and RIF1 (RAP1-interacting factor 1 homolog), lack enzymatic activities directly implicated in DNA repair, we decided to further explore the 53BP1-dependent pathway. In this study, we uncovered a nuclease, Artemis, as a PTIP-binding protein. Loss of Artemis restores PARPi resistance in BRCA1-deficient cells. Collectively, our data demonstrate that Artemis is the major downstream effector of the 53BP1 pathway, which prevents end resection and promotes nonhomologous end-joining and therefore directly competes with the homologous recombination repair pathway

PTIP associates with Artemis to dictate DNA repair pathway choice

PARP inhibitors (PARPis) are being used in patients with BRCA1/2 mutations. However, doubly deficient BRCA1−/−53BP1−/− cells or tumors become resistant to PARPis. Since 53BP1 or its known downstream effectors, PTIP and RIF1 (RAP1-interacting factor 1 homolog), lack enzymatic activities directly implicated in DNA repair, we decided to further explore the 53BP1-dependent pathway. In this study, we uncovered a nuclease, Artemis, as a PTIP-binding protein. Loss of Artemis restores PARPi resistance in BRCA1-deficient cells. Collectively, our data demonstrate that Artemis is the major downstream effector of the 53BP1 pathway, which prevents end resection and promotes nonhomologous end-joining and therefore directly competes with the homologous recombination repair pathway